A novel psychrotrophic fungus, <Emphasis Type="Italic">Mortierella minutissima</Emphasis>, for <Emphasis Type="BoldSmallCaps">D</Emphasis>-limonene biotransformation

Of 98 strains of moulds, isolated from arctic soils, Mortierella minutissima 01, grew the best on agar plates with limonene vapor. Perillyl alcohol and perillic acid were the main products of limonene biotransformation. Maximal yield of perillyl alcohol (125mgl^–1) occurred in medium containing 0.8% substrate, at 15°C, pH 6 and after 4–5 d. Revisions requested 27 October 2004; Revisions received 27 November 2004     

The Heat Shock Gene, <Emphasis Type="Italic">htpG</Emphasis>, and Thermotolerance in the Cyanobacterium, <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The htpG null mutant was obtained by inserting a chloramphenicol resistance cassette (Cm ^r) in the htpG coding sequence. The htpG null mutant (htpG), hsp16.6, and the double mutant, htpG::hsp16.6 cells showed little growth disadvantage at 30°C and 37°C, but not at 40°C. This suggests that HtpG and HSP16.6 proteins do not have an essential role during growth at normal and mildly elevated temperatures. Cell growth, cell survival rate, and oxygen electrode measurements demonstrated that htpG, hsp16.6, and htpG::hsp16.6 cells were sensitive to heat stress. Decreased basal and acquired thermotolerance was observed when mutants were heat shocked, with htpG::hsp16.6 being the most sensitive. A comparison of mutants showed that hsp16.6 was more sensitive to heat shock than htpG. Received: 19 November 2002 / Accepted: 19 December 2002     

Expression of <Emphasis Type="Italic">cspH</Emphasis> upon nutrient up-shift in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium

The gene cspH , which encodes one of the cold-shock proteins in Salmonella enterica serovar Typhimurium, has previously been reported to be induced during early exponential phase at 37°C. In the present study, the expression of cspH upon nutrient up-shift at 37°C was investigated and found to be affected by DNA gyrase and DNA-binding protein Fis. When cells at stationary phase were subcultured into a rich medium, the mRNA level of cspH increased dramatically prior to the first cell division. However, when the cells were treated with DNA gyrase inhibitors, cspH mRNA was not induced upon nutrient up-shift. The low level of DNA superhelical density at the cspH promoter in part affected the expression of cspH mRNA in vitro. In addition, a fis-deficient strain had a lower level of cspH mRNA than the wild-type upon nutrient up-shift. Finally, a cspH–lacZ construct, in which the putative binding region for Fis was deleted in the cspH promoter, expressed a low level of LacZ, in contrast to the native cspH–lacZ construct.This revised version was published online in July 2004 with corrections to Fig. 4.     

A review of the culture potential of <Emphasis Type="Italic">Solea solea</Emphasis> and <Emphasis Type="Italic">S. senegalensis</Emphasis>

A number of scientific studies have investigated aspects of soles(Solea soleaandS. senegalensis) ecology, population genetics and biology in their natural environment, and the species have been extensively studied in captivity during the last decade. Studies on the genetic population structure of sole indicate that several distinct breeding populations exist within its distributional range in European waters. Recent studies suggest a phylogenetic relatedness ofS. soleaand S. senegalensis, being found as closest sister lineages in most reconstructions. However, studies on molecular genetics and morphological traits give diagnostic differences that consistently lead to their taxonomic separation at the specific rank. Studies show that sole spawn readily in captivity, and the buoyant, fertilized eggs are easily collected. Stocking density during maturation should be 1–1.5kg/m², and temperature should be kept above 16°C (S. senegalensis) or between 8 and 12°C (S. solea). In nature, the onset of spawning is related to a rise in temperature occurring during spring (March–June). Salinity should be kept constant around 33–35 and the fish reared under simulated natural photoperiod (LDN). In other cultured flatfish species, a change in the photoperiod is the key environmental signal used to manipulate and control maturation, but at present time there are no published work that verifies or contradicts this for either S. senegalensisor S. solea. Studies indicate that a mixture of inert and live food may increase the weaning success of sole fry, and this can be further enhanced by using attractants in the dry feed. Future experiments are needed to determine the ideal time to commence weaning and determine the minimum duration of this period. Studies on alternative feeding strategies are also required. The effect of temperature and photoperiod on juvenile growth has not been studied systematically in neither of the two species and the relative importance of a direct photoperiod effect on growth in sole therefore remains to be defined.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected.     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> bulblet regeneration from immature embryos of endangered <Emphasis Type="Italic">Sternbergia fischeriana</Emphasis>

Sternbergia fischeriana is an endangered geophyte and therefore in vitro micropropagation of this plant will have great importance for germplasm conservation and commercial production. Bulb scale and immature embryo explants of S. fischeriana were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. Immature embryos produced higher number of bulblets than bulb scales. Large numbers of bulblets were regenerated (over 80 bulblets/explants) from immature embryos on Murashige and Skoog (MS) medium supplemented with 4 mg l^–1 6-benzylaminopurine (BA) and 0.25 mg l^–1 -naphthaleneacetic (NAA) or 2 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D) after 14 months of culture initiation. Regenerated bulblets were kept at 5 °C for 5 weeks and then transplanted to a potting mixture.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Effects of growth temperature upshift on cell cycle progression in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cell cycle progression of Cryptococcus neoformans was studied for cells grown exponentially at 15°, 24°, and 30°C. Except for speed, cell cycle progression was similar. In particular, budding occurred relatively soon after initiation of DNA synthesis at 15°, 24°, and 30°C. After growth temperature was shifted from 15° to 30°C, cells were transiently arrested before initiation of DNA synthesis. Thus, similar to Saccharomyces erevisiae, Start was the main susceptible cell cycle controlling point in C. neoformans. However, after spontaneous release from arrest as above, cells were further arrested in the unbudded state. Thus, the timing of budding was delayed just before the G₂ phase, or even until after entering the G₂ phase, but it was also transient, and 5h after the shift buds emerged relatively soon after initiation of DNA synthesis. Thus, C. neoformans cells can respond adaptively to mild stress by delaying budding. The existence of the second susceptible cell cycle control point, i.e., budding, appears to endow C. neoformans with a unique characteristic of stronger inhibition of multiplication than growth. A model of the C. neoformans cell cycle is also presented.     

Probiotic properties of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains isolated from porcine gastrointestinal tract

One strain of Lactobacillus salivarius, two strains of Lactobacillus reuteri and Lactobacillus amylovorus, and two strains of Bifidobacterium thermacidophilum with antagonistic effect against Clostridium perfringens were isolated from porcine gastrointestinal tract. Isolates were assayed for their ability to survive in synthetic gastric juice at pH 2.5 and were examined for their ability to grow on agar plate containing porcine bile extract. There was a large variation in the survival of the isolates in gastric juice and growth in the medium containing 0.3% (w/v) bile. L. salivarius G11 and L. amylovorus S6 adhered to the HT-29 epithelial cell line. Cell-free supernatant of L. amylovorus S6 showed higher antagonistic activity as effective as the antibiotics such as neomycin, chlortetracycline, and oxytetracycline against bacterial pathogens including C. perfringens, Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae, Edwardsiella tarda, and Aeromonas salmonicida subsp. salmonicida.     

Psychrophilic <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> requires <Emphasis Type="Italic">trans</Emphasis>-monounsaturated fatty acid for growth at higher temperature

A psychrophilic bacterium, Pseudomonas syringae (Lz4W) from Antarctica, was used as a model system to establish a correlation, if any, between thermal adaptation, trans-fatty acid content and membrane fluidity. In addition, attempts were made to clone and sequence the cti gene of P. syringae (Lz4W) so as to establish its characteristics with respect to the cti of other Pseudomonas spp. and also to in vitro mutagenize the cti gene so as to generate a cti null mutant. The bacterium showed increased proportion of saturated and trans-monounsaturated fatty acids when grown at 28°C compared to cells grown at 5°C, and the membrane fluidity decreased with growth temperature. In the mutant, the trans-fatty acid was not synthesized, and the membrane fluidity also decreased with growth temperature, but the decrease was not to the extent that was observed in the wild-type cells. Thus, it would appear that synthesis of trans-fatty acid and modulation of membrane fluidity to levels comparable to the wild-type cells is essential for growth at higher temperatures since the mutant exhibits growth arrest at 28°C. In fact, the cti null mutant-complemented strain of P. syringae (Lz4W-C30b) that was capable of synthesizing the trans-fatty acid was indeed capable of growth at 28°C, thus confirming the above contention. The cti gene of P. syringae (Lz4W) that was cloned and sequenced exhibited high sequence identity with the cti of other Pseudomonas spp. and exhibited all the conserved features.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Infections of the Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

We report the first systematic survey for the presence of Wolbachia endosymbionts in aphids and whiteflies, particularly different populations and biotypes of Bemisia tabaci. Additional agriculturally important species included were predator species, leafhoppers, and lepidopterans. We used a polymerase chain reaction (PCR)-based detection assay with ribosomal 16S rDNA and Wolbachia cell surface protein (wsp) gene primers. Wolbachia were detected in a number of whitefly populations and species, whitefly predators, and one leafhopper species; however, none of the aphid species tested were found infected. Single, double, and triple infections were detected in some of the B. tabaci populations. PCR and phylogenetic analysis of wsp gene sequences indicated that all Wolbachia strains found belong to group B. Topologies of the optimal tree derived by maximum likelihood (ML) and a ML tree in which Wolbachia sequences from B. tabaci are constrained to be monophyletic are significantly different. Our results indicate that there have been at least four independent Wolbachia infection events in B. tabaci. The importance of the presence of Wolbachia infections in B. tabaci is discussed. RID= ID= <E5>Correspondence to: </E5>K. Bourtzis; <E5>email:</E5> kbourtz&commat;cc.uoi.gr Received: 9 September 2002 / Accepted: 25 September 2002     

Heterospecific Expression of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Cell Shape Determination Genes <Emphasis Type="Italic">mreBCD</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>*

The divIVB operon of Bacillus subtilis includes the cell shape-associated mre genes, including the membrane-associated proteins MreC and MreD. TnphoA mutagenesis was utilized to analyze a topological model for MreC. MreC has a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large carboxy terminal domain which lies externally to the outer leaflet of the cell membrane. Expression of the B. subtilis MreB protein, or the Mre C and D proteins, results in a morphological conversion of the Escherichia coli host cells from a rod to a roughly spherical cell, morphologically similar to mre-negative mutants of E. coli. Immunolocalization of the MreC protein in B. subtilis revealed that this protein is found at the midcell division site of the bacterial cells, consistent with the postulated role of the Mre proteins in the regulation of septum-specific peptidoglycan synthesis. RID= ID= <E5>Correspondence to: </E5>G.C. Stewart; <E5>email:</E5> stewart&commat;vet.ksu.edu Received: 5 August 2002 / Accepted: 7 October 2002     

Regulation of the <Emphasis Type="Italic">htpX</Emphasis> Gene of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> and Its Expression in <Emphasis Type="Italic">E. coli</Emphasis>

Xylella fastidiosa was the first phytopathogen to be completely sequenced, and its genome revealed several interesting features to be used in functional studies. In the present work, the htpX gene, which encodes a protein involved in the heat shock response in other bacteria, was analyzed by RT-PCR by using cells derived from different cultural conditions. This gene was induced after a temperature upshift to 37°C after growth in minimal medium, XDM, but showed constitutive expression in rich medium or in XDM plus plant extracts. Sequences upstream to the htpX gene, containing a putative regulatory region, were also transferred to E. coli, and the thermoregulation was maintained in the new host, since it was constitutively transcribed at 37°C or 45°C in all culture media tested, but not at 28°C in minimal culture medium. The gene was also cloned into the expression vector pET32Xa/LIC, and the expression of the corresponding protein was confirmed by Western blotting.     

In vitro selection of resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella</Emphasis> spp. at <Emphasis Type="Italic">in vivo</Emphasis> fluoroquinolone concentrations

Background  

Fluoroquinolones are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections. However, resistance to fluoroquinolones in Enterobacteriaceae is increasingly reported. Studies assessing the ability of fluoroquinolones to select for resistance have often used antimicrobial concentrations quite different from those actually acquired at the site of infection. The present study compared the ability to select for resistance of levofloxacin, ciprofloxacin and prulifloxacin at concentrations observed in vivo in twenty strains of Escherichia coli and Klebsiella spp. isolated from patients with respiratory and urinary infections. The frequencies of spontaneous single-step mutations at plasma peak and trough antibiotic concentrations were calculated. Multi-step selection of resistance was evaluated by performing 10 serial cultures on agar plates containing a linear gradient from trough to peak antimicrobial concentrations, followed by 10 subcultures on antibiotic-free agar. E. coli resistant strains selected after multi-step selection were characterized for DNA mutations by sequencing gyrA, gyrB, parC and parE genes.     

<Emphasis Type="Italic">tdd8</Emphasis>: a TerD domain-encoding gene involved in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> differentiation

The Streptomyces coelicolor genome contains 17 TerD domain-encoding genes (tdd genes) of unknown function. The proteins encoded by these genes have been presumed to be involved in tellurite resistance on the basis of their homology with the protein TerD of Serratia marcescens. To elucidate the role of a Tdd protein (Tdd8), both a deletion mutant for the corresponding gene tdd8 (SCO2368) and a recombinant strain over-expressing tdd8 were produced in S. coelicolor M145. The deletion mutant (Δtdd8), like the wild strain, was not resistant to potassium tellurite. The deletion was not lethal but had a marked effect on differentiation. The deletion strain showed more rapid growth in liquid medium and produced long chains of short spores with a dense and non-spherical spore wall on agar plates. The strain over-expressing tdd8 had a growth delay in liquid medium and produced very few spores of irregular shapes and sizes on solid medium. The results of this study demonstrated that Tdd proteins might have a function other than tellurite resistance and this function seems to be of crucial importance for the proper development of the actinomycete S. coelicolor.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Replication and Host Cell Division in <Emphasis Type="Italic">Aedes albopictus</Emphasis>

Wolbachia pipientis is an obligate intracellular endosymbiont of a range of arthropod species. The microbe is best known for its manipulations of host reproduction that include inducing cytoplasmic incompatibility, parthenogenesis, feminization, and male-killing. Like other vertically transmitted intracellular symbionts, Wolbachias replication rate must not outpace that of its host cells if it is to remain benign. The mosquito Aedes albopictus is naturally infected both singly and doubly with different strains of Wolbachia pipientis. During diapause in mosquito eggs, no host cell division is believed to occur. Further development is triggered only by subsequent exposure of the egg to water. This study uses diapause in Wolbachia-infected Aedes albopictus eggs to determine whether symbiont replication slows or stops when host cell division ceases or whether it continues at a low but constant rate. We have shown that Wolbachia densities in eggs are greatest during embryonation and then decline throughout diapause, suggesting that Wolbachia replication is dependent on host cell replication.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.